Identification of Proteins Affecting Assembly, Regulation, and Function of Thylakoid Protein Complexes

影响类囊体蛋白复合物组装、调节和功能的蛋白质的鉴定

• 批准号: 9728400
• 负责人: Willem Vermaas
• 金额: $ 30万
• 依托单位: Arizona State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-15 至 2001-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9728400&HistoricalAwards=false
• 关键词: Identification; Proteins; Affecting; Assembly; Regulation

This research project will utilize the genomic sequence of the cyanobacterium Synechocystis sp. PCC 6803 (accessible through http://www.kazusa.or.jp/cyano/cyano.html) to functionally identify "new" proteins involved in the regulation, assembly and function of protein complexes that catalyze electron transfer in thylakoid membranes of this cyanobacterium. This project involves positive selection for spontaneous mutants (1) in which photosystem II activity is decreased or abolished, or (2) in which regulation of expression of particular genes has been modified. Positive selection will occur by screening for high-light tolerance of the Synechocystis sp. PCC 6803 strain that lacks photosystem I; this strain is sensitive to high light but can be propagated at high light intensity if photosystem II is impaired or if rates of electron transport processes in the thylakoid membrane or the regulation by particular redox components have been altered.The availability of the genomic DNA sequence of Synechocystis sp. PCC 6803 provides a powerful approach to a rapid identification of the location of spontaneous mutations that lead to high-light tolerance of the photosystem I-less strain. DNA from these positively selected mutants will be fragmented by restriction enzymes; each digest (treated with one enzyme) will be size-separated on a gel and gel slices (each reflecting a certain size range of DNA) will be tested for functional complementation of the original (high-light sensitive) strain from which the spontaneous mutant was derived. A complete restriction map of the Synechocystis sp. PCC 6803 genome has been constructed for 16 enzymes that cut this cyanobacterial genome 300-1000 times each. For each of the 16 restriction enzymes, the resulting fragments, identified through their genomic position, have been sorted by size and compiled into a table. Based on the size range of the fragments that can functionally complement for each restriction enzyme, an appropriate size range of restriction fragments can be selected from each table. These restriction fragments (each identified through their position on the genome) are pooled, and are then sorted according to the genomic nucleotide number at the end of each fragment. Upon inspection of this sorted list, clusters of nucleotide numbers can be identified. Generally only a single cluster can be found in which there is one representative from each of the selected size regions for each of the enzymes. By identifying the region that is common between all sequences in this cluster, the domain carrying the mutation of interest (generally not longer than 2 kbp) can be determined. The domain of interest will be amplified by PCR, the mutation will be determined, and mutations in unknown genes can thus be simply mapped and identified, even without cloning.Some of the complementing secondary mutations may be in known genes, but others will be in open reading frames for which no function is known yet. These open reading frames will be of particular interest as the results obtained will help to assign a role to these known sequences with unknown function. This method already has led to the identification of an open reading frame that is similar to ycf39 from chloroplasts and that affects photosystem II function in mutants. The next part of the project will be the preliminary analysis of the role of open reading frames that were identified. The types of analysis will depend on the open reading frame and on the phenotype of the mutant, but may involve the following: (1) sequence homology searches, (2) functional analysis of electron transport, (3) gene expression studies, and (4) protein synthesis and degradation measurements.This work will lead to the identification of currently unknown genes whose products may be involved in photosystem II assembly, in electron transfer out of the plastoquinone pool in thylakoid membranes, or in control of regulatory processes in the cell. This is an open field, but rapid progress will be made now the genomic sequencing of Synechocystis sp. PCC 6803 has been completed, enabling effective and comprehensive means of analysis such as the one described here. This area of research not only is an important complement to large-scale sequencing efforts, in that it provides information regarding the function of open reading frames, but also is expected to provide novel insights into the regulation mechanisms and assembly processes required for electron transport in thylakoid membranes.

该研究项目将利用蓝细菌合成细菌的基因组序列。 PCC 6803（可通过http://www.kazusa.or..jp/cyano/cyano.html访问），以便在功能上识别参与蛋白质复合物的调节，组装和功能的“新”蛋白质，这些蛋白质复合物的催化电子蛋白质复合物催化了这种cyanyakoid membranes中的电子转移。该项目涉及对自发突变体的正选择（1），其中光系统II活性降低或废除，或者（2），其中已修改了特定基因表达的调节。阳性选择将通过筛选出现synechocystis sp的高光耐受性。 PCC 6803缺乏光系统i；该菌株对高光很敏感，但如果光明系统II受到损害，或者如果类囊体膜中电子传输过程的速率或特定的氧化还原组件的调节，则可以在高光强度繁殖。 PCC 6803提供了一种强大的方法，可以快速识别自发突变的位置，从而导致光系统I-i-nele-nele-necl菌株的高光耐受性。这些积极选择的突变体中的DNA将被限制酶分散。每种摘要（用一种酶处理）将在凝胶和凝胶切片上进行大小分离（每个反映一定尺寸的DNA范围），将测试以对自发突变体的原始（高光敏）菌株的功能互补进行测试。 Synechocystis sp的完整限制图。 PCC 6803基因组是为16种酶构建的，该酶每次切割该蓝细菌基因组300-1000倍。对于16种限制酶中的每一个，通过其基因组位置识别的所得片段已按大小进行排序并编译成表。基于可以在每个限制酶功能补充的片段的尺寸范围内，可以从每个表中选择适当的限制片段范围。这些限制片段（每个片段通过其在基因组上的位置鉴定）被合并，然后根据每个片段末端的基因组核苷酸数进行分类。在检查此排序列表后，可以识别核苷酸数的簇。通常，只能找到一个单个簇，其中每个酶的每个尺寸区域都有一个代表。通过识别该群集中所有序列之间常见的区域，可以确定带有感兴趣突变的域（通常不超过2 kbp）。感兴趣的领域将通过PCR扩大，将确定突变，即使没有克隆即使在未知基因中的突变也可以简单地映射和鉴定。一种补充的二次突变可能是在已知的基因中，但其他基因可能会在开放的阅读框架中，尚未知道该功能。这些开放式阅读框将特别感兴趣，因为获得的结果将有助于为这些已知序列分配一个未知功能的序列。该方法已经导致了与叶绿体类似的开放阅读框架的鉴定，并影响突变体中的光系统II功能。该项目的下一部分将是对所确定的开放阅读框架作用的初步分析。 The types of analysis will depend on the open reading frame and on the phenotype of the mutant, but may involve the following: (1) sequence homology searches, (2) functional analysis of electron transport, (3) gene expression studies, and (4) protein synthesis and degradation measurements.This work will lead to the identification of currently unknown genes whose products may be involved in photosystem II assembly, in electron transfer out of the plastoquinone pool在类囊体膜中，或控制细胞中的调节过程。这是一个开放的场，但是现在将在Synechocystis sp的基因组测序中取得快速进步。 PCC 6803已经完成，具有有效而全面的分析方式，例如此处描述的一种。这一研究领域不仅是大规模测序工作的重要补充，因为它提供了有关开放阅读框架功能的信息，而且还有望提供有关囊体膜中电子传输所需的调节机制和组装过程的新见解。